Defining the Role of Boi1/Boi2 in the Coordination of Actin Polarity and Polarized Exocytosis. Cell polarity is critical for many eukaryotic cellular processes (e.g. migration, cell growth, etc.). Since many of the factors involved in polarity are conserved, the budding yeast Saccharomyces cerevisiae are a valuable system to study the basic mechanisms underlying this process. Rho GTPases control cell polarity via the separate regulation and coordination of the actin polarity and secretory pathways; however, the mechanisms involved are still unclear. This proposal aims to characterize a means by which Rho proteins can coordinate these two pathways. Bem One Interacting proteins Boi1 and Boi2 and their orthologs have been implicated in cell polarity under the regulation of Rho3, Cdc42, and phospholipids. These proteins are functionally redundant and essential for polarized growth, but their exact function and direct regulation by Rho proteins is still undetermined. To better understand Boi proteins' regulation, the first aim will identify the Rho-binding element and test their binding specificities for all five yeast Rho GTPases using in vitro binding assays, and examine the effects of mutations in the Rho-binding element on Boi function in polarized growth using genetic assays. These experiments will specify which Rho GTPases regulate Boi proteins and distinguish Boi regulation by Rho proteins from phospholipids. The role of Boi1 and Boi2 in actin polarity is undetermined, though studies in other systems suggest that Boi proteins mediate formin activation to polarize the actin cytoskeleton. To determine if this role in budding yeast is conserved, the second aim will test for physical interactions between Boi and formin proteins using the in vitro binding system and test the significance of this interaction using genetic analyses and immunofluorescence. Preliminary data reveals a novel actin-independent role for Boi proteins in polarized secretion. To begin to define this role, the third aim will examine Boi proteins' interactions with the exocytic machinery using co-immunoprecipitation and Western analysis and use genetics to determine the significance of these interactions and how Rho proteins and phospholipids regulate Boi proteins' role in secretion. Together these aims will provide a better understanding of the separate regulation of polarized secretion and begin to elucidate how secretion contributes to overall cell polarity.